The analysis of the human genome is expected to be substantially completed in the early twenty-first century. For effective utilization of the outcome of the analysis, it is indispensable to develop a new technology for artificially manipulating nucleic acids (the carrying, sequence recognition, and on-off action of transcription or translation of nucleic acids). The most important material for manipulating nucleic acids is considered to be a carrier capable of supporting or carrying nucleic acids such as DNA, i.e. a gene vector. However, in-vivo use of conventional gene vectors composed of artificial materials have produced no significant results in human clinical studies. This can be attributed especially to low gene-transferring efficiency, limited expression of gene information (Cotton et al., Meth. Enzymol. 217: 618-644 (1993)), or insufficient biocompatibility of cationic carrier materials (Choksakulnimitr et al., J. Control. Rel., 32: 233-241 (1995)).
Although viruses such as retroviruses (Miller, Nature 357: 445-460 (1992)) or adenoviruses (Mulligan, Science 260: 926-932 (1993)) have shown very promising in-vitro results as gene vectors, in-vivo use of these naturally occurring materials is restricted, especially because of inflammatory action, immunogenicitic properties or the risk of integration into the genome of the viruses or mutagenesis induction due to the viruses (Crystal, Science 270: 404-410 (1995)).
Thus, as a substitute for such naturally-originating gene vectors, there has been proposed use of non-viral vectors composed of an artificial material which can be handled in an easier manner and can carry DNAs into the cells in a more efficient manner as compared with the viral vectors (Tomlinson and Rolland, J. Contr. Res., 39: 357-372 (1996)).
As materials for the transfection there have been proposed synthetic vectors based on water-soluble cationic polymers, i.e., based on the “lipofection” using cationic lipids (Gao and Huang, Gene Therapy 2: 710-722 (1995)) or amphipathic substances (Behr, Bioconjugate Chem. 5: 382-389 (1994)) Examples include the use of poly (L-lysine) (PLL) (Wu and Wu, Biotherapy 3: 87-95 (1991), DEAE-dextran (Gopal, Mol. Cell. Biol. 5: 1183-1195 (1985)), dendrimers (Haensler and Szoka, Bioconjugate Chem. 4: 372-379 (1993), or cationic methacrlylate derivatives (Wolfert et al., Hum. Gene Ther. 7: 2123-2133 (1996)). The decisive advantage of “polyfection” in which a cationic polymer is used is that almost infinite structural variations are possible which may affect physicochemical and biological properties of the polymer and that the polymer can form a complex with a plasmid. Thus, the vectors can be efficiently used when bound to cell-specific ligands such as transferrin (Qagner et al., Proc. Natl. Acad. Sci. 87: 3410-3414 (1991)), asialoglycoproteins (Wu and Wu, J. Bio. Chem. 262: 4429-4432 (1987)), antibodies (Trubetskoy et al., Bioconjugate Chem. 3: 323-327 (1992)), or carbohydrates (Midoux et al., Nucleic Acid Research 21: 871-878 (1993)).
At present, it is polyethyleneimine (PEI) that is the most extensively studied as a non-viral, artificial vector. It has been shown that PEI, a cationic polymer assuming a three dimensional branched structure in various adhesive and floating cells, may result in tranfection in a considerably highly efficient manner (Boussif et al., Gene Therapy 3: 1074-1080 (1996)). For example, 95% of in-vitro transfection with the 3T3 fibroblast cell line was accomplished. In-vivo gene transfection into mouse brain using PEI as a mediator resulted in long-term expressions of the reporter gene and Bcl 2 gene in the neuron and the glial cell, the results being comparable to those obtained with the gene transfection using the adenovirus (Aldallah et al., Hum. Gene Ther. 7: 1947-1954 (1996)).
However, the safety of cationic polymers such as polyethylimine has not yet been confirmed. While the presence of amino groups is indispensable so as to impart a cationic charge to such polymer, an amino group has a risk of toxicity in the body due to its high physiological activity. In fact, no cationic polymer studied so far has yet been put into practice, or yet been registered in the “Pharmaceutical Additives Handbook” (edited by the Pharmaceutical Additives Association of Japan and published by Yakujinipposha Publishing Co.).
Extensive studies have also been made on compounds capable of recognizing a nucleic acid sequence, such as peptide-bound nucleic acid compounds, calicheamicins (an antibiotic) and DNA-bound proteins. In addition, rapid progress has been made recently in the studies on the recognition of genes and the control of translation of genetic information by using artificial proteins. While the studies on compounds capable of interacting with nucleic acids such as DNA and RNA are being conducted as an important subject, most of the studies are directed toward unessential side issues. Few studies are found which are made based on fundamental subjects, for example, on what types of materials (compounds) will interact with a nucleic acid in general. For example, cationic materials such as polyethyleneimine, on which the current studies are being focused, are not suitable for use as an agent for carrying genes, in consideration of the facts that (1) they combine with a nucleic acid to form a complex which, once formed, will not readily decompose because of bonding due to electrostatic interaction, (2) that they are toxic, and (3) that the polycation will react with the phosphoric acid (which makes the nucleic acid water-soluble) to form a complex which is usually non-soluble. In addition, most of the compounds conventionally known undergo an irreversible reaction as an intercalater resulting in the destruction of nucleic acids (genetic information).
It is an object of the present invention to provide a new type of gene manipulator (gene carrier) which is capable of interacting with and bonding to a nucleic acid such as DNA and RNA, without destroying the nucleic acid, to form a water-soluble complex so as to be applicable under biological conditions, and which is also capable of dissociating the nucleic acid from the complex and rebonding to such nucleic acid when necessary.